Project Summary Objectives: The long-term objective of this application is to develop novel assays, panels, and kits to enable rapid and early point-of-care (POC) testing of TB in children. The envisioned POC test would be administered in home by mothers to their children, and in local healthcare centers by community healthcare workers who can diagnose and begin treatment of children with TB within minutes upon first presentation, instead of hours or days. The POC test will use an innovative biotechnology called, Isothermal Chain Reaction (ICR). ICR is a disruptive and enabling assay technology platform that will offer a new class of reagents relevant to TB medical healthcare and biomedical research. ICR does not require any enzymes, primers, nucleotides, master mixes, extension reactions, cDNA intermediates, or thermocycling. These benefits overcome constraints of PCR- based technologies by being resistant to enzyme inhibitors found in laboratory samples and clinical specimens, having higher specificity for rare mutations, and lowering reagent costs. ICR is anticipated to enable routine TB detection in children in a more sensitive, selective, rapid, and affordable manner, without having to invest in costly instruments and labor-intensive sample preparation procedures for testing in local community settings. Specific Aims: 1. Development of ICR probes to TB target sequences: We will design and develop ICR assays to rapidly and isothermally detect synthetic target sequences associated with TB in a mixed population of templates in less than 10 minutes under isothermal conditions, without enzymes, primers, nucleotides, or master mixes. 2. Detection of TB in non-sputum samples: We will use ICR assays from Aim 1 to detect TB in retrospective exhaled breath condensation, saliva, and urine samples. Results will be benchmarked to mycobacterial growth indicator tube cultures to demonstrate the feasibility of ICR TB detection in non-sputum samples. 3. Detection of paucibacillary forms of TB from pleural fluid samples: We will use selected assays as in Aim 2 to detect TB in retrospective pleural fluid samples. Results will be benchmarked and compared to PCR to demonstrate the feasibility that ICR has enhanced selectivity to detect TB pathogens missed by PCR in the paucibacillary form of TB common in children, and that ICR is more amenable to POC TB testing. Impact: Accomplishing the Specific Aims could significantly impact rapid and early detection of TB pathogens in children when therapeutic interventions are much more effective, and enable routine POC testing to screen, monitor, treat, and stop transmission of childhood TB in local community healthcare settings.